Analytical validation of frataxin proteoforms in blood as biomarkers of Friedreich's ataxia

血液中 frataxin 蛋白质形式作为弗里德赖希共济失调生物标志物的分析验证

• 批准号: 10117295
• 负责人: Ian Alexander Blair
• 金额: $ 75.4万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-15 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10117295
• 关键词: Address; Affect; Age; Alleles; Alternative Splicing; Amino Acids; Biological; Biological Assay; Biological Markers; Blood; Blood Cells; Blood Platelets; Blood specimen; Cardiac; Cell Nucleus; Cessation of life; Clinical; Collection; Cultured Cells; Cytosol; Development; Disease; E protein; Erythrocytes; Friedreich Ataxia; Genes; Half-Life; High Pressure Liquid Chromatography; Immunoassay; Lateral; Length; Life; Mass Spectrum Analysis; Measurement; Methionine; Methods; Mitochondria; Mitochondrial Proteins; Monitor; Mutation; N-terminal; Pathology; Patients; Peptides; Plasma; Protein Isoforms; Proteins; Reaction; Resolution; Sampling; Sensitivity and Specificity; Serum; Site; Source; Specificity; Testing; Therapeutic Intervention; Therapeutic Trials; Time; Treatment Efficacy; Triplet Multiple Birth; Up-Regulation; Validation; Whole Blood; base; frataxin; healthy volunteer; liquid chromatography mass spectrometry; mitochondrial processing peptidase; nano; novel; novel therapeutic intervention; sample collection; stable isotope; therapy design; two-dimensional

ABSTRACT: ANALYTICAL VALIDATION OF FRATAXIN PROTEOFORMS IN BLOOD AS BIOMARKERS OF FRIEDREICH’S ATAXIA Friedreich’s ataxia (FA) is an autosomal recessive disease caused by an intronic GAA triplet expansion in the FXN gene, leading to reduced expression of the mitochondrial protein frataxin. FA is estimated to affect 1 in 50,000 with a mean age of death in the fourth decade of life. There are no approved treatments for FA, although experimental approaches, which involve up-regulation or replacement of frataxin protein, are being tested. Frataxin is undetectable in serum or plasma, and whole blood could not be used because frataxin was thought to be present in long-lived erythrocytes. An assay for analyzing frataxin in platelets, which have a half- life of 10 days, which would allow therapeutic interventions to be tested, was developed by the Blair and Lynch labs. The assay is based on stable isotope dilution immunopurification two-dimensional nano-ultra high performance liquid chromatography/parallel reaction monitoring/high resolution mass spectrometry (nano- UPLC-MS/HRMS) and is monitoring three tryptic peptides from frataxin. The assay had 100 % sensitivity and specificity for discriminating between controls and FA cases but analyzing platelets on a routine basis is very challenging in many clinical settings. We have now discovered that in erythrocyte is in fact a novel proteoform of frataxin (isoform E) with 135-amino acids (76-210) and an N-terminally acetylated methionine residue. It arises through an alternative splice-site form that is used for the canonical full-length form of frataxin (1-210). There is three times as much isoform E in erythrocytes (26.7 ± 6.4 ng/mL) from the blood of healthy volunteers (n=10) when compared with the mature mitochondrial frataxin present in other blood cells (7.1 ± 1.0 ng/mL). We recently found that both isoform E (8.5 ± 1.1 ng/mL) and mature mitochondrial frataxin (2.1 ± 1.1 ng/mL) are both reduced by > 70 % in blood from FA patients (n=29) when compared with healthy control subjects. Isoform E lacks a mitochondrial targeting sequence and so it is distributed to both cytosol and the nucleus when expressed in cultured cells. The ability to specifically quantify extra-mitochondrial and mitochondrial isoforms of frataxin in whole blood would make it possible to follow the progress of the disease and monitor the efficacy of therapeutic interventions. This will require the development of rigorously validated assays that address the pre-analytical and analytical issues that are relevant to the use of blood as a biological matrix for biomarker analysis rather than the more common use of serum or plasma. The present proposal addresses these issues under following specific aims: Aim 1: To conduct pre-analytical validation for collection and storage of mature frataxin and isoform E protein in whole blood. Aim 2: To conduct analytical validation of the method for quantifying mature mitochondrial frataxin and frataxin isoform E in whole blood. Aim 3: Validation of the mature frataxin and frataxin isoform E proteoform analysis in whole blood from controls, FA carriers, and FA cases from multiple sites.

文摘：血液中Frataxin蛋白作为生物标志物的分析验证 弗里德里希共济失调 Friedreich‘s共济失调(FA)是一种常染色体隐性遗传病，由内含子GAA三联体扩增引起 FXN基因，导致线粒体蛋白Frataxin表达减少。据估计，FA将影响1/3 5万人，平均死亡年龄在生命的第四个十年。目前还没有批准的治疗FA的方法， 尽管涉及上调或替代Frataxin蛋白的实验方法正在进行中 测试过。在血清或血浆中检测不到Frataxin，而且不能使用全血，因为Frataxin是 被认为存在于长寿的红细胞中。一种分析血小板中Frataxin的分析方法，血小板中有一半- 布莱尔和林奇夫妇开发了10天的寿命，这将允许测试治疗干预措施 实验室。该检测是基于稳定同位素稀释免疫纯化的二维纳米超高 高效液相色谱/平行反应监测/高分辨质谱仪(纳米级 UPLC-MS/HRMS)，并正在监测来自Frataxin的三种胰蛋白酶多肽。该方法具有100%的灵敏度和较高的灵敏度。 区分对照组和FA病例的特异性，但常规分析血小板非常 在许多临床环境中具有挑战性。我们现在已经发现，在红细胞中实际上是一种新的蛋白质形式 由135个氨基酸(76-210)和N-末端乙酰化蛋氨酸残基组成的Frataxin(异构体E)。它 通过用于Frataxin(1-210)的标准全长形式的另一种剪接位点形式产生。 健康志愿者血液中红细胞中的异构体E含量(26.7±6.4 ng/mL)是正常人的三倍 (n=10)与其他血细胞中存在的成熟线粒体Frataxin(7.1±1.0 ng/mL)相比。 我们最近发现，异构体E(8.5±1.1 ng/mL)和成熟线粒体Frataxin(2.1±1.1 ng/mL) 与健康对照组相比，FA患者(n=29)的血液中这两种物质的含量都降低了70%。 异构体E缺乏线粒体靶向序列，因此它既分布在细胞质中，也分布在细胞核中 当在培养的细胞中表达时。专门量化线粒体外和线粒体的能力 全血中的Frataxin亚型将使跟踪疾病的进展和监测 治疗性干预的有效性。这将需要开发经过严格验证的分析方法， 解决与使用血液作为生物基质相关的分析前和分析问题 生物标志物分析，而不是更常见的血清或血浆使用。本提案涉及 这些问题的具体目标如下： 目的1：对成熟Frataxin及其异构体E蛋白的收集和储存进行分析前验证 全血的。目的2：对成熟线粒体定量方法进行分析验证 全血中的Frataxin和Frataxin亚型E。目的3：验证成熟的Frataxin和Frataxin亚型E 对照组、FA携带者和多个部位FA患者全血中的蛋白质形态分析。